In vertebrates and invertebrates, Bone Morphogenetic Proteins (BMPs) act in a morphogen gradient to pattern the dorsoventral (DV) axis in a concentration dependent manner. The shape of the BMP morphogen gradient is critical: precise amounts of BMP signaling at discrete DV positions specify distinct cell fates. In the zebrafish embryo, high BMP signaling specifies ventral cell fates, intermediate levels specify lateral fates, and no BMP signaling results in dorsal fates. The BMP signaling gradient shape is continually regulated from blastula through gastrula stages, as DV tissues are progressively patterned from anterior to posterior. Since BMPs also pattern the neural tube and developing limbs, and act in the progression of cancer, osteoporosis, osteoarthritis, fibrodysplasia ossificans progressiva, understanding the spatiotemporal mechanisms that shape the BMP gradient in DV patterning will provide paradigms for BMP function and its modulation in these other contexts. The goal of this proposal is to determine how the BMP signaling gradient is shaped during gastrulation. We can determine the shape of the BMP signaling gradient through a quantitative immunofluorescence assay of nuclear phosphorylated Smad1/5, a direct readout of active BMP signaling. The initial BMP signaling gradient is generated at mid-blastula stages by Chordin (Chd), a key extracellular antagonist that binds and inhibits BMP ligands from signaling. Importantly, we observe that the gradient shape at the start of gastrulation differs fro its shape during late gastrulation. Since Chd activity is required throughout gastrulation, it is vtal to determine how Chd is regulated in time and space. Chd activity is modulated by two key classes of proteins: (i) the homologous metalloproteases Bmp1a and Tolloid (Tld), which cleave and inactivate Chd, and (ii) the metalloprotease inhibitor Sizzled (Szl). How loss of function of both metalloproteases affects the BMP gradient spatially and temporally will be investigated. Additionally, how loss of Szl function affects the gradient will be examined. It is hypothesized that Bmp1a/Tld inhibit Chd without restraint early in gastrulation to generate the BMP signaling gradient, while Szl is critical to inhibit Bmp1a/Tld during the latter half of gastrulation to steeen the BMP signaling gradient. In this proposal, the temporal requirement for Bmp1a/Tld and Szl, how each protein affects the shape of the BMP signaling gradient, and the effective ranges of Tld and Szl protein activity will be determined. Together, these experiments will elucidate how the spatiotemporal regulation of metalloprotease activity correctly shapes the BMP signaling gradient to pattern the DV axis of the developing zebrafish embryo.